Next generation sequencing (NGS) techniques provide the means for rapid and cost-effective analysis with an unprecedented level of detail and depth. However, a current problem with the application of NGS platforms is that true low frequency variants often cannot be effectively distinguished from detected variants caused by errors during sample preparation or sequencing. For example, the bases in DNA fragments can undergo chemical modifications during isolation, fixation, storage or processing. These modifications can alter the way that DNA modifying enzymes interact with the DNA fragments. An example of such a modification is the oxidation of a cytosine residue. Once oxidized, a DNA polymerase will no longer recognize this base as a “C” nucleotide; it now recognizes this as a U. In the process of determining the base sequence of this DNA fragment, it is typical for a DNA polymerase to make a copy of the fragment in the library generation process. When the sequence of this fragment is determined, there will be an A/T base pair in place of the original G/C base pair. It will appear as though this fragment had a Single Nucleotide Variant (SNV) in this location. Since the original fragment did not have this variant, it will be a false positive modification. Other fragments representing the same genomic location will not have this variant. However, it would not be possible to tell if this was a processing error or a very low frequency event.
Unambiguous low frequency event determination is particularly important in analyzing materials obtained from liquid biopsy samples. Liquid biopsies are analysis of materials extracted from biological fluids (e.g., blood or plasma). It is hoped that indication of disease can be detected in these samples so more invasive sampling can be avoided. DNA from dead or lysed cells are the typical analytes. Since disease cells make up a very small proportion of total cells within the body, DNA from these cells makes up a very small amount of the DNA present in these biological fluids. Since the consequence of a false positive modification can result in the necessity to obtain an invasive sample for verification, methods for reducing false positive results for low frequency events are needed.